A transient disruption of fibroblastic transcriptional regulatory network facilitatestrans-differentiation

Abstract

Transcriptional Regulatory Networks (TRNs) coordinate multiple transcription factors (TFs) in concert to maintain tissue homeostasis and cellular function. The re-establishment of target cell TRNs has been previously implicated in direct trans-differentiation studies where the newly introduced TFs switch on a set of key regulatory factors to induce de novo expression and function. However, the extent to which TRNs in starting cell types, such as dermal fibroblasts, protect cells from undergoing cellular reprogramming remains largely unexplored. In order to identify TFs specific to maintaining the fibroblast state, we performed systematic knockdown of 18 fibroblast-enriched TFs and analyzed differential mRNA expression against the same 18 genes, building a Matrix-RNAi. The resulting expression matrix revealed seven highly interconnected TFs. Interestingly, suppressing four out of seven TFs generated lipid droplets and induced PPARG and CEBPA expression in the presence of adipocyte-inducing medium only, while negative control knockdown cells maintained fibroblastic character in the same induction regime. Global gene expression analyses further revealed that the knockdown-induced adipocytes expressed genes associated with lipid metabolism and significantly suppressed fibroblast genes. Overall, this study reveals the critical role of the TRN in protecting cells against aberrant reprogramming, and demonstrates the vulnerability of donor cell's TRNs, offering a novel strategy to induce transgene-free trans-differentiations.

Regulatory network-based imputation of dropouts in single-cell RNA sequencing data

“…S8). Interestingly, TWIST2 and PRRX1, described by Tomaru et al 18 to interact with OSR1, also showed fibroblast-specific expression ( Fig. S9).…”

Gatekeeper transcription factors regulate switch between lineage preservation and cell plasticity

“…Studies have shown that in intermediately transformed cells, some specific genes of the starting cells are persistently expressed, whose siRNA mediated knockdown considerably improved reprogramming efficiency (Ebrahimi, 2015;Mikkelsen et al, 2008). Though multiple gene knockdown of the starting fibroblast cell's Transcriptional Regulatory Network (TRN) yielded differentiated adipocytes (Tomaru et al, 2014) this concept however was never quite extended to deciphering the impact of repression of a single critical lineage-instructive TF of the starting cell in affecting transdifferentiation. Similarly, while TFs that interfere with dedifferentiation have been demonstrated to induce cell-type-specific transcriptional profiles, repression of the identified TFs in prior studies were not successful in permitting dedifferentiation (Hikichi et al, 2013), likely because of differences in the employed siRNA transfection protocol.…”

Engineering Cell Fate: The Roles of iPSC Transcription Factors, Chemicals, Barriers and Enhancing Factors in Reprogramming and Transdifferentiation

“…Notably, Ding et al speculated that transient expression of iPSC-TFs during cell activation phase erases the epigenetic identity of the examined (starting) cells [62,64]. Interestingly, Tomaru and colleagues have recently shown that knockdown of fibroblast transcriptional regulatory networks (TRNs) in human fibroblast cells followed by a specific chemical/cytokine cocktail can lead to transdifferentiation of fibroblasts without expression of any transgene [196]. Suggestively, disruption of the somatic gene regulatory network may erase starting cell identity and cause an unstable or plastic state similar to the action of iPSC-TFs in the CASD paradigm and may provide a novel strategy for enhancing pluripotent reprogramming and transdifferentiation [196].…”

“…Interestingly, Tomaru and colleagues have recently shown that knockdown of fibroblast transcriptional regulatory networks (TRNs) in human fibroblast cells followed by a specific chemical/cytokine cocktail can lead to transdifferentiation of fibroblasts without expression of any transgene [196]. Suggestively, disruption of the somatic gene regulatory network may erase starting cell identity and cause an unstable or plastic state similar to the action of iPSC-TFs in the CASD paradigm and may provide a novel strategy for enhancing pluripotent reprogramming and transdifferentiation [196]. Thus, removing cell identity by selective knockdown of somatic master genes and pushing the cells in any direction by specific soluble signals offer ideas for the production of various cell types without counting on ectopic expression of virus-mediated transgenes.…”